ES2250119T3 - PROCEDURES AND MEANS FOR EXTRACORPORAL ORGAN PERFUSION. - Google Patents

Abstract

Apparatus and methods of operation of apparatus for the extracorporeal perfusion of organs are provided which support viability and function of an organ such as a liver, generally outside the body. Blood is supplied as described herein to an organ such as a liver under physiological pressure but without forcing blood flow at any particular rate such that the organ is allowed to auto-regulate blood flow. This allows for organ preservation or resuscitation prior to transplantation, maintenance of organs for use in experimental study of isolated liver physiology, and treatment of patients suffering from organ failure.

Description

Procedures and means for infusion extracorporeal organs.

The present invention relates to perfusion of organs, in particular extracorporeal organ perfusion, whether human or nonhuman, for example pigs, and whether they are not transgenic or transgenic. Device and procedures are available of operation of said apparatus for infusion to give support for the viability and function of an organ such as the liver, Usually out of body. This allows conservation or Resurrection of the organ before a transplant, for example while the recipient of a transplant is prepared, maintaining organs for use in an experimental study of physiology isolated liver, and the treatment of patients suffering from a organic dysfunction

The invention is based on the results Unexpected inventors related to self-regulation of blood flow in an organ. Advantageously, according to the present invention blood is provided to an organ, such as a liver, under physiological pressure but without forcing blood flow at any particular speed. The organ is allowed to self-regulate blood flow. (You can afford to leave any excess blood flow.) Also advantageously, it can maintain the outlet pressure of a perfused organ (vena cava in a liver). Histological appearances of perfused livers using the invention are included within the normal limits after 72 hours. This represents an advance important regarding known techniques, whose problems date from a long time ago.

Perfusion of the isolated liver has been studied for more than 90 years (Bernard C., CR Acad. Sci. 1855; 41: 461). However, much work has been carried out on perfusion of the experimental isolated liver with non-blood solutions in rodent models in order to evaluate different aspects of liver function (Gores et al., Hepatology, 1986; 6: 511-7). The clinical application of ex vivo liver perfusion as a means to support patients with acute liver failure was thoroughly studied in the late 1960s and early 1970s (Eiseman et al., Annals of Surgery, 1965, 329-345 ; Eiseman, Ann. Roy. Coll. Surg. Engl. 1965; 38: 329-349; Watts et al., British Medical Journal, 1967; 341-345; Abouna, The Lancet, 1968.1216-1218; Abouna and others , Brit. J. Surg., 1969; 56: 223-225; Condon et al., The American Journal of Surgery, 1970; 119: 147-154; Abouna others, Lancet, 1970; 391-396; Lempinen et al., Scandinavian Journal of Gastroenterology, 1971; 6: 377-383; Chalstrey et al., British Medical Journal, 1971; 58: 522-524; Hickman et al., Scand. J. Gastroent., 1971; 6: 563-568; Parbhoo et al. , Lancet, 1971; 659-665; Abouna et al., Surgery, 1972; 71: 537-546; Abouna et al., Surgery Obstetrics and Gynaecology, 1973; 137: 741-752; Abouna et al., Transplantation, 1974; 18: 395-408). However, this technique fell into disuse between the late seventies and early nineties, due to the appearance of other treatments, particularly the successful liver transplant.

More recently there has been a resurgence of interest in extracorporeal liver perfusion (Fair et al., AASLD, 1993, A899; Fox et al., American Journal of Gastroenterology, 1993; 88: 1876-1881; Chari et al., NEJM, 1994; 331: 234-237). This has led to improvements in cardiopulmonary bypass technology, which has also led to successful extracorporeal membrane oxygenation, and genetic engineering developments, which has resulted in the production of transgenic pigs resistant to hyperacute rejection (Cozzi and others, Nature Medicine, 1995; 1: 964-966). The genetic modification of the regulation of complement activation has nullified the immediate immunological reaction between previously discordant species.

Although the use of organs of concordant species It has greater immunological advantages, there are important practical restrictions. The availability of human livers is limited and those that are available should be used for the clinical transplant The use of non-human primate organs poses important problems, although baboon organs have been used successfully. There is a greater concern in relation to zoonosis of primates; also, most nonhuman primates do not achieve the necessary size to be effective in replacement of the human organ.

It is known that the critical effector mechanism in the hyperacute rejection among discordant species is the activation of the complement waterfall (Platt and others: Role of Natural Antibody-Antigen in Xenotransplantation. In: Xenotransplantation Cooper and others, eds. Berlin: Springer, 1997; 17-23). Complement activation normally occurs. suppressed by the presence of cell surface proteins species-specific, regulators of the activity of the complement (Liszewski et al., Adv. Immunol. 1996; 61: 201-283). Recently, a pig colony transgenic for one of these regulators has produced the factor of the acceleration of human decay (hDAF) (Cozzi and White, Nat. Med. 1995; 1: 964-966). There are indications that hearts and kidneys of these animals, transplanted in primates, they are not rejected in a hyper manner and behave like if it were a harmonious species (Schmoeckel and others, Transplant Proc. 1997; 29: 3157-3158). Thus, transgenic pigs provide a source of organs "concordant".

These factors have stimulated various groups to re-explore the possibility of supporting patients with liver failure with extracorporeal porcine livers. He Organ transplant success has also stimulated interest in the use of extracorporeal perfusion as a means of conservation and resurrection of organs from marginal donors (Schon et al., 8th Congress of the European Society for Organ Transplantation, 1997, Abstract TRP4).

An important limitation of previous studies It has been the short duration of the infusion, no more than 24 hours (Neuhaus et al., Int. J. Artif. Organs, 1993; 16: 729-739).

WO99 / 15011 describes a device designed specifically for use in the infusion of a heart, and circuits adapted for use with other organs: kidney, liver, pancreas and lungs The devices proposed for the kidney, the liver or lungs are not able to allow any self-regulation of the flow by the organ. Figure 8 and the text that accompanies, for example, proposes an apparatus for infusion of liver in which the fluid is going to be pumped in and out at the same pressure, forcing the organ to take the blood that is provides The proposed cardiac apparatus (figure 5) would be inappropriate for use in any other organ. Working normal, blood is pumped to the heart and there is no possibility of self-regulation, by the heart, of the flow through the same. Optionally, when the system is to be used for perfusion of a beating heart (not a hot heart, no latiente), the authors use an excess from the entrance to the aorta. In an optional embodiment of WO99 / 15011, if left beat a heart, although the document does not describe it, a heart in that situation would have allowed him to self-regulate blood flow to through it. This optional embodiment in WO99 / 15011 represents an accidental anticipation that has been waived since the claim 1 according to G2 / 03.

US5807737 describes a perfusion apparatus of the heart that controls and forces the maintenance of pressure. He heart must take all the forced pressure towards it through of the pumped circuit.

EP0376763 describes a perfusion apparatus in the that the blood flow is pumped to the organ and the blood flow goes out directly to a container. The pump imposes the inflow towards the organ while the blood pressure presented to organ will vary with the internal resistance of the organ.

In one aspect, the present invention provides a mammalian organ perfusion apparatus which, when connected to the entry artery and exit vein of a mammalian organ, selected from the group consisting of liver, kidney, pancreas, and small intestine provides a fluid circuit for flow blood through the organ, in whose circuit it exists, preferably in the following order, an output channel for the connection to the exit vein of the organ, an outlet pump of adjustable speed to maintain the physiological pressure in the vein output of the organ, an oxygenator and a heat exchanger, and an input channel for connection to an input artery of the organ, the circuit also comprising a device for maintain physiological pressure and variable flow in the artery of organ input while allowing the organ to self-regulate the blood flow through itself to achieve blood flow physiological.

In another aspect where the organ is a liver, The present invention provides a liver perfusion apparatus of mammals that, when connected to the vena cava, the hepatic artery and the portal vein of a mammalian liver provides a circuit of fluid for blood flow through the liver, in whose circuit there is, preferably in the following order, an output channel for connection to the vena cava, a speed output pump adjustable to maintain the physiological pressure in the vena cava, a oxygenator and a heat exchanger, and a fork channel which divides the circuit to the input channels for connection to the hepatic artery and portal vein, comprising the circuit, in addition, a device to maintain the physiological pressure in the hepatic artery and physiological flow in the portal vein while the liver is allowed, when connected to the device, self-regulate blood flow through the artery and self-regulate the pressure in the portal vein.

The said device maintains a circuit for the Blood flow. An individual can join the circuit, preferably a human. The individual's circulation can connect to the circuit through cannulas arranged in the major veins (placed through open surgery or percutaneously). The individual's outflow is returned to the Perfusion circuit at a point before the organ. The entrance to individual is of blood that has passed through the organ and has oxygenated. As an example with reference to the device circuit of perfusion shown in figure 1, the connection to a patient can be at a point after the clamp of gate indicated in the branch shown in the figures leading to the container, with the return from the patient leading to the vessel. As indicated, organ support Extracorporeal of humans has been practiced for many years, techniques and techniques are well known to those skilled in the art precautions to be taken in connecting patients to extracorporeal perfusion apparatus. For example, it is normal supply prostacyclin and heparin, and allow to evacuate bile of the liver A circuit may additionally include one or more flow meters and one or more pressure meters.

Whereas previously the infusion extracorporeal or organs such as livers have been based on a arbitrary selection of flow or pressure (in the portal vein for a liver), these have not varied independently.

The present invention allows the maintenance of physiological levels of both flow and pressure. The inventors have observed that in the liver the portal pressure is related directly with the pressure of the inferior vena cava and that the flow portal (instead of pressure) is directly related to the pressure applied from the portal venous vessel (in embodiments preferred portal venous vessel height above the liver where blood is supplied from there by gravity). The liver responds to the increase in portal pressure by reducing the portal vascular resistance, maintaining portal pressure constant.

In the circuit of the invention, the flow of organ outlet is pumped to maintain pressure physiological, for example varying the pumping speed, in the vein relevant, which for the liver is vena cava. Input flow physiological can be achieved by adjusting the pressure of supply, for example, the portal vein for the liver. In a preferred embodiment of the present invention, the portal flow physiological is obtained by adjusting the height of the portal vessel on the liver. The present invention allows establishment of normal levels not only of portal pressure but also portal flow. The flow of arterial input to a liver perfused according to the invention can be set to a physiological pressure, determining the flow rate by means of the vascular resistance of the liver.

The general principle of the invention, as applicable to a variety of organs, is to set the pressure of the input flow at a physiological level, with a flow rate variable that depends on the vascular resistance of the organ, allowing physiological self-regulation to occur.

The circuit used in aspects of the invention as exemplified by the liver, it also includes a device to maintain the physiological pressure in the artery liver and physiological flow in the portal vein while allows the liver, when connected to the device, self-regulate blood flow through the artery and self-regulate the pressure in the portal vein. Said device may include a container for collecting excess blood resulting from the self-regulation by the liver flow in the hepatic artery, and said container may be for the blood supply to the channel of entrance for the connection to the portal vein of the liver. Supply of blood to the portal vein of a liver when connected to the device It can be from the container under the force of gravity.

Conveniently, the inlet flow pressure can be regulated while allowing self-regulation of the inlet flow rate by arranging a fork tube that allows evacuation from the connection to the entrance to the vessel blood The resistance of the evacuation bypass of the bifurcation can be altered by a partial occlusion, by example by tightening, for adjusting the pressure at the inlet organ. The self-regulation of the flow by the organ leads to a evacuation of excess blood to the container. Alternative modes of get the same result include an automated controller that set the pressure within predetermined parameters but that allow variable flow in response to self-regulation by organ. In the case of a liver, said system can use two pumps, one to provide a hepatic arterial flow under pressure constant / variable flow, and one to fill the venous vessel low pressure portal.

In another aspect, the present invention provides a procedure for operating an organ perfusion apparatus. He The device can have the components identified here. A performing a procedure in accordance with this aspect of the invention applied to a liver can be a procedure that makes operate the apparatus for perfusion of a mammalian liver connected to the vena cava, the hepatic artery and the portal vein of a mammalian liver in which the apparatus provides a circuit of fluid for blood flow through the liver, including the procedure regulate the pumping rate of the outflow to maintain the physiological pressure in the vena cava, regulate supply pressure to the inlet channel connected to the artery liver to maintain physiological pressure in the hepatic artery and physiological flow in the portal vein, and allow the liver self-regulate blood flow through the artery and self-regulate the pressure in the portal vein.

Another aspect provides a procedure of extracorporeally perfusion of a mammalian organ, the organ other than a heart that is allowed to beat, whose procedure includes pumping arterial blood flow from entrance to the organ to maintain physiological pressure and flow variable in the artery of the organ and pumping venous flow of exit from the organ to maintain the physiological pressure in the exit vein of the organ, while allowing the organ self-regulate blood flow through yourself to get a physiological arterial flow. The organ can be selected from group consisting of liver, kidney, pancreas and intestine thin.

If the organ is a liver, a procedure of according to the invention may include the regulation of the flow of the portal vein and pumping blood flow out of the liver to maintain physiological pressure in the vena cava of the liver, while The liver is allowed to self-regulate portal pressure.

Another aspect of the invention provides a procedure to maintain the viability of a mammalian organ, in whose procedure the organ is perfused according to a procedure as described. The perfusion according with the present invention allows the maintenance of viability of an organ such as a liver for at least about 72 hours, preferably at least about 96 hours, more preferably at least about 120 hours.

The experimental exemplification of embodiments of the present invention included below refers to perfusion of livers. In the light of the results of the inventors, it is scientifically reasonable to assume that the apparatus and The methods of the invention are effective when applied to other organs, such as kidneys, pancreas or small intestine. The perfusion of an organ with a single entry (such as a kidney) it will imply a modified circuit with the return of the container towards pump inlet Excess blood flow can provide the organ artery with a fixed pressure while outlet means are provided for any blood flow that is is in excess of that required by the organ. Alternatively, arterial flow can be provided by of a pump at a variable flow rate with a fixed blood pressure.

An perfused organ according to the present invention can be human or non-human, and can be non-transgenic or transgenic. Non-human organs can be modified to increase compatibility with connection to a human and / or perfusion with human blood. An example of a modification that is successfully used to increase the concordance of non-human organs, especially porcine organs, is transgenic modification, such that the organs express the acceleration factor of the decay of the human complement component (hDAF) (see, for example, Cozzi and White, and Schmoeckel and others, supra ).

The blood used in the infusion can be human or non-human, for example pig for a pig organ, and This may depend on the purpose of the infusion.

A human organ will usually be perfused with human blood. If the organ is going to connect to a human subject, the blood will be human and will need to be antigenically compatible with ABO. A non-human organ, for example a Pork organ, can be perfused with relevant blood not human (for example, pig), or human blood for example if the organ is modified in the manner described above (for example, transgenic for hDAF).

Preferably the blood is citrated. The inventors have found that the function of perfused livers with citrated blood is markedly superior to those perfused with heparinized blood Citrate is a metabolic substrate important liver.

In a liver perfusion circuit according with the invention, the fluid that escapes from the organ, including the ascites, preferably circulated back to the liver, preferably through the portal vein or, if not, through the Hepatic artery. This has the advantage of keeping the volume vascular, and also to retain plasma proteins that they would probably be lost if ascites were replaced by colloid / crystalloid solutions.

The apparatus according to the invention can include a channel for collecting fluid that escapes from the organ and transport to the incoming blood vessel.

Preferably the infusion is a "perfusion hot ", that is, at a physiological temperature, usually 37 ° C for a human organ and 39 ° C for a pig organ.

Preferably, a liver for use in the The present invention is preserved with hypertonic citrate, instead of the current preservation solution for normal liver transplantation (University of Wisconsin) based on lactobionate. The solution of preservation is rinsed out of the liver immediately before Start the infusion.

Another advantage of a perfusion circuit of according to the present invention derives from the fact that when said circuit is connected to a patient, the patient's circulation It connects to the circuit vessel and, therefore, the speed The patient's perfusion may be different from the infusion extracorporeal organs.

The inventors have also found that perfusion of a pig liver, whether normal or genetically modified, with human blood results in the extraction of human red blood cells by the Kupffer cells of the liver. For clinical application purposes, blocking this function of Kupffer cells, for example by ablation of the cells, allows long-term perfusion without excessive consumption of red blood cells. The function of Kupffer cells can be blocked / ablated by the treatment of the donor animal before organ removal or by treatment of the organ with clodrinate or gadolinium, or immunoglobulin or immunoglobulin / complement lysis, or using any appropriate available technology. for those skilled in the art.

In accordance with another aspect of this invention, a method is provided to reduce the consumption of red blood cells by a non-human organ in perfusion with blood human, the procedure comprising the application to the organ of a blocking or ablation treatment of cell function Kupffer before exposure of the organ to human circulation or perfusion with human blood, in which a technique of perfusion in accordance with other aspects of this invention.

Other aspects and embodiments of this invention will be clear to those skilled in the art in view of the experimental exemplification that follows with reference to the figures.

Brief description of the figures

Figure 1 illustrates a perfusion apparatus of according to an embodiment of the invention, connected to a liver.

Figure 2 shows the hemodynamic parameters during extracorporeal liver perfusion as described in Example 1 that follows:

Figure 2a shows the total flow as measured leaving the liver through the inferior vena cava, and the IVC pressure measured in the confluence of hepatic veins.

Figure 2b shows the portal flow as measured by entering the liver through the portal vein, and the portal pressure measured by controlling the line in The portal vein.

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Figure 2c shows the flow of the hepatic artery calculated as the difference between total and portal flow, and hepatic artery pressure measured as direct arterial line pressure.

Figure 3 shows the metabolic parameters during extracorporeal liver perfusion as described in Example 1 measured by arterial blood gasometry (CIBA Corning, 288 Blood Gas System),

Figure 3a shows the normal reference pH range from 7.35 to 7.45, and the base excess of -2 to 2 mEq.

Figure 3b shows the normal reference potassium range of 3.5 to 4.6 mmol / L, and ionized calcium of 1.13 to 1.3 mmol / L.

Figure 4 shows function indicators synthetic during extracorporeal liver perfusion.

Figure 4a shows urea levels versus creatinine levels expressed on axis scales of clinically ordered relevant.

Figure 4b shows the activity of the complement measured by CH50 analysis using an RBC sheep. The data is simply expressed as a percentage of the CH50 units of the plasma just before the liver perfusion

Figure 4c shows factor V levels measured by replacing human blood deficient in factor V and expressed as a percentage of normal human factor V levels.

Figure 5 shows indicators of injury and liver function during extracorporeal perfusion of the liver.

Figure 5a shows ALT levels (SGPT) (normal human margin up to 50 U) and alkaline phosphatase levels (normal human margin 30-135 U).

Figure 5b shows the production of bile measured as ml of bile produced by hour and total bilirubin expressed in \ mumol / L (human margin normal up to 17 µm / L).

Figure 5c shows the oxygen consumption calculated by the Fick equation and expressed as ml O2 / min / liver.

Figure 6 shows metabolic parameters during extracorporeal liver perfusion as described in Example 2 as measured by blood gasometry arterial.

Figure 6a shows the normal human reference margin for excess base which is -2.5 to 2.5 mEq.

Figure 6b shows the normal human reference margin for potassium which is of 3.5 to 4.6 mmol / L.

Figure 7 shows the production of measured bile as ml of bile produced per hour and total bilirubin expressed in µmol / L (normal human range up to 17 µm / L).

Figure 7a shows a blood-perfused hDAF transgenic pig liver human

Figure 7b shows a non-transgenic swine liver perfused with blood human

Figure 7c shows a non-transgenic swine liver perfused with blood swine

Figure 8 shows a complete blood count that is performed using a Coulter counter (T660) regulated for swine blood measurement. Transgenic represents pig livers HDAF transgenic perfused with human blood, Non-transgenic represents wild pig livers perfused with human blood and alloperfusion represents wild pig pigs perfused with swine blood

Figure 8a shows a platelet count, (X 10 9 / L).

Figure 8b shows nucleated cell counts (X109 / L).

Figure 8c hematocrit sample (expressed as a percentage of hematocrit of blood flow before starting the infusion extracorporeal).

Figure 9a urea sample measured through a clinical laboratory technique normal. Transgenic versus Normal, p = NS.

The figure that 9b shows the activity of the complement measured using an analysis functional by lysis of sheep red blood cells and the determination of CH50 The results are expressed as percentage of CH50 present before perfusion of the liver (primer blood). Transgenic versus Normal, p = NS.

Figure 9c shows the activity of factor V measured using an analysis functional by reconstitution of human serum deficient in factor V. The results are expressed as percentage of human serum normal. Transgenic versus Normal, p = NS.

Figure 10 shows an excess base during the liver support and rescue, indicating rapid rejection after Let the infusion cease.

Example 1 Extracorporeal perfusion of pig livers Blood donor

In these studies white pigs were used large (35 - 70 kg), five as liver donors and five as blood donors All animals were treated according with the Animal Protection Act., 1986 from the United Kingdom. Pigs blood donors were previously medicated with 10 mg / kg of ketamine (Willows Francis Veterinary, Ketaset) and 1 mg / kg of midazolam (Roche, Hypnovel) IM, followed by halothane anesthesia. The External jugular vein was cannulated, 15,000 U of intravenous heparin and blood was collected in bags cited by gravity drainage until circulation ceased. Blood was used immediately or stored at 4 ° C and used Within a week of collection.

Donor liver

The animals were previously medicated as indicated above, a vein in the ear was cannulated and anesthesia was induced using 4 mg / kg of propofol intravenously (Zeneca, Diprivan). The animal was tubed using an extended endotracheal tube. Pulse oximetry was used through a tail probe to follow oxygenation and end-tidal CO2 was measured. After intubation, a 14-gauge cannula was placed to allow fluid replacement during hepatectomy. Anesthesia was maintained with propofol IV (10 mg / kg / hr) throughout the procedure. In the midline an incision was made. The bile duct was divided and the hepatic vessels were identified and isolated in a standard way. The liver was dissected until it was connected to the donor by its vascular fixations. Throughout this procedure, meticulous attention was paid to hemostasis to minimize subsequent blood loss in the heparinized extracorporeal circuit. 20,000 units of heparin were given intravenously and allowed to circulate. The infrarenal aorta was cannulated with a 20-gauge cannula (Bard), and was connected to a closed system that contained a cold Eurocollins solution (Baxter, Soltran,). The portal vein was cannulated with a 24-gauge cannula (Bard) in a similar manner and the infusion began with a cold Eurocollins solution through the portal vein and the aorta by gravity as the suprahepatic inferior vena cava (IVC) becomes divided into the pericardium.

After the infusion of 2 liters of solution Cold Eurocollins through the liver, the liver was extracted removing a diaphragm cut around the suprahepatic IVC, dividing the hepatic artery in the celiac axis, the IVC infrahepatic at the level of the renal veins, and the portal vein at the level of the splenic vein. While the infusion continued portal, the liver was extracted from the animal and was arranged in a low temperature container. The diaphragmatic remnant was Sutured with prolene 3.0 to ensure hemostasis.

The suprahepatic IVC was cannulated with a cannula 28 gauge (Bard) with its hole positioned at the level of the hepatic veins The inferior vena cava was cannulated with a cannula pressure control Fr (Portex) as well as the portal vein to through a tributary. The hepatic artery was cannulated with a cannula 10 Fr (Jostra). Arterial pressures were measured directly from the arterial limb of the circuit. The control Continuous pressure was achieved using a transducer (Viggo-Spectramed, Haemodynamic Monitoring Set) and a digital monitor (Datex, AS / 3). The bile duct was cannulated with a 14-gauge silastic T-tube with the open end of the T-tube placed in a collection device to control the bile production

Perfusion Circuit

While the preparation of the liver bank work, the perfusion apparatus (see Figure 1) it was mounted and given a layer of 1,500 c.c. pig's blood donor. The perfusion circuit consisted of an oxygenator (Jostra M15 pediatric membrane oxygenator), a centrifugal pump (Medtronic BP50 centrifugal pump), a container (container of Jostra soft structure 800 ml), tubes (Medtronic, PVC, 1/4 and 3/16 inch inside diameter (ID) with Medtronic connectors polycarbonate), a gate clamp, transducers pressure (Baxter triple pressure transducers) and flow probes (2 Medtronic DP 38P). The oxygenator was connected to an exchanger of heat to maintain the blood temperature at 39 ° C (normal temperature for a pig).

During the preparation of the circuit the pH, paCO 2 and Ca 2+ so that they were within the range normal physiological In general, the perfusion circuit was completed with 9.2 mmol of CaCl2, 20 mmol of sodium bicarbonate and 7,000 heparin units. Once the blood was optimized in the circuit, then a sample was obtained for a complete blood count, electrolytes, urea, creatinine, liver function tests (LFT), total bilirubin, and plasma was collected and stored in Liquid nitrogen for other analyzes.

Before the liver connection to the circuit perfusion, the Eurocollins solution was rinsed from the portal circulation using 1 liter of infusion solution colloidal (Behring, Haemaccel). This also allowed a complete exclusion of air from the liver and cannulas. The liver was arranged in an intestinal pouch and then suspended in physiological serum in a sterile perfusion chamber. Two plastic tubes were placed soft in the most dependent part of the intestinal bag. These tubes were then connected to the vessel through a pump Imed (Imed, Gemini PC-4) to recirculate any ascites produced. The liver was connected to the perfusion device primed preventing air remaining (figure 1).

The infusion was executed at a speed of 1-2 liters / min (total) with 100-400 ml / min through the hepatic artery. Blood pressure is maintained at 80-100 mmHg (average) and the IVC pressure at 0-3 mmHg. The IVC and arterial pressure are regulated through pump speed and portal flow to through the height of the container.

Liver perfusion

After the start of the infusion, they were obtained arterial and venous blood samples at 1 min, 5 min, 10 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, and then every 4 hours until end of the infusion. Blood samples were analyzed. immediately by a blood gas analysis (CIBA Corning, 288 Blood Gas System). The oxygen flow and the air flow at oxygenator were regulated to maintain the paO2 between 14-20 kPa and the paCO_2 between 3.5-6 kPa

Blood samples were then analyzed for a complete blood count, electrolytes, urea, creatinine, LFTs and Total bilirubin. The plasma was stored in liquid nitrogen for a subsequent analysis of complement and coagulation components.

The oxygen consumption was calculated using the Fick's principle using the following equation:

Contained in O 2 = 1.39 ([Hgb] in g / dl) (O 2 in%) + 0.0031 (pO2)

Each gram of hemoglobin is capable of transporting 1.39 ml of O2 and the amount of dissolved O2 is a function Linear PO2 (0.0031 ml / dl blood / mm HgPO2). Establish The oxygen consumption of the liver is complicated due to the fact That the liver has a portal blood supply. Therefore it is it is necessary to know the O2 content of the hepatic artery, the Portal vein and hepatic vein. In this isolated liver system, the PaO 2 of the portal vein and the hepatic artery is the same and for consequently the oxygen consumption of the liver can be calculated as follow:

Consume of O2 = (PO2hepatic artery - PO2VCI) (Flow Total)

Immediately before infusion of the liver with blood, an infusion of prostacyclin (Glaxo Wellcome, Flolan) at 4 mg / hr was initiated; In addition, an infusion of heparin at 500 U / hr was performed and regulated to maintain ACT> 300 sec. Once the liver perfusion began, parenteral nutrition was provided. An infusion of a mixture containing 500 ml of a standard total parenteral nutrition solution (Kabi Pharmacia), 50 ml of a 10% lipid emulsion (Clintec, IVELIP), 10 ml of trace elements (Pharmacia & Upjohn, Additrace, was made ), 1 vial of multivitamins (Clintec, Cernevit), and 72 units of insulin (Novo Nordisk Pharmaceuticals, Ltd., Human Actrapid) at 7 ml / hr during the entire 72-hour infusion. Additional glucose or insulin was provided to keep glucose within a range of 4-10 mmol / l as determined through a blood glucose stick analysis (Boehringer-Mannheim machine, Accutrend and BM-Accutest sticks ). At the beginning of the infusion and every 24 hours after it, 1 g of cefotaxime (Roussel, Claforan) was added to the circuit.

The perfusion of the liver ceased electively to 72 hours and liver samples were sent for evaluation histological Random samples of the liver were cut and they froze for an immunohistochemical evaluation and they looked at formalin for staining hematoxylin and eosin.

Sample Analysis

Serum samples from pig sera were analyzed for complement production by CH50 analysis (Harrison R. Complement technology. In: Weir D. M., Herzenberg L. A., Blackwell C., eds. Handbook or Experimental Immunology, 4th Edition, Oxford: Blackwell Scientific Publications, 1986: 39.1-39.49). Briefly, red blood cells were washed sheep (SRBC) three times in a fixative diluent complement (ICN Biomedicals. Inc.), diluted by 10%, and coated with SO-16 antibody for 30 minutes in ice. 25 µL of a coated SRBC solution was added with 1% SO-16 to each cavity of a plate 96 cavity microtiter. Serum samples were examined. from variable time points during liver perfusion starting with a 1:10 dilution and serially diluted to 1: 4 by tripled through the microtiter plate. The values of CH50 were calculated as described by Harrison (Harrison R. Complement Technology In: Weir D. M., Herzenberg, L.A., Blackwell C., eds. Handbook of Experimental Inmunolgy, 4th Edition, Oxford: Blackwell Scientific Publications, 1986: 39.1-39.49). There was a variability in values of CH50 in the primer blood (pig blood before perfusion through the liver) between experiments but values consistent in the experiments; therefore all Values are expressed as a percentage of primer.

Factor V levels were measured using an Organon Teknika MD180 machine and Organon consumables for a PT-based factor V test (to nullify the effects of anticoagulation of heparin). Prothrombin time is measured using the same technique. The electrolytes, the LFTs, the Total bilirubin, creatinine, and urea were measured using standard clinical procedures. Hemograms complete including hemoglobin, hematocrit, blood cell count white, and platelet count were obtained using a counter Coulter (T660) regulated for the measurement of swine blood.

Results

In five experiments, which lasted 72 hours each one, the average total hepatic flow was 1.99 l / min (s. 0.21) at an average IVC pressure of 2.06 mmHg (s. d. 3.0) (Figure 2A). He mean portal flow was 1.75 l / min (s. d. 0.21) at a pressure mean of 7.22 mm / Hg (s. d. 2.3) (Figure 2B). Mean arterial flow it was 0.24 l / min (s. d. 0.18) at an average pressure of 90.3 mm / Hg (s. d. 9.5) (Figure 2C).

The metabolic function was evaluated by measuring the pH, HCO 3-, K +, excess base and oxygen consumption. He pH control, HCO 3-, and excess base demonstrated essentially the normal acid base balance throughout the perfusion (figure 3A). Similarly, the K + remained in physiological levels throughout the infusion (figure 3B).

As an index of synthetic liver function, it evaluated the levels of urea, complement and factor V and the Results are illustrated in Figures 4A, 4B and 4C. Urea level The mean for the 5 experiments was 2.92 mmol / L (s. d. 0.55) at beginning of perfusion and 45.4 mmol / L (s. 6.0) at the end. This is in contrast to the creatinine level that increased from 100 mmol / L (s. d 18.32) to 136.8 mmol / L (s. d. 36.22). This implies that the increase in urea is the result of synthesis, unlike Hemoconcentration There was a progressive increase in complement activity such that for 72 hours it was found in a average of 383.2% of the initial level (s. d. 111.1). Instead, Factor V levels remained relatively constant, starting with an average level of 408.4% (s. 125.5 d) and ending with an amount similar to 72 hours of 343% (s. 171.5 d).

Liver injury was assessed by measuring the Alanine transferase (ALT) level of liver enzymes and alkaline phosphatase (Alk. Phos.). After 72 hours of infusion the ALT level had varied from 62.4 units / L (s. d. 10.1) to 51.4 units / L (s. d 8.9) (figure 5A). Alkaline phosphatase levels fell during the perfusion period of 136.5 units / liter (s. d. 17.0) at 70.2 units / liter (d. 12.9) at 72 hours. The levels of bilirubin remained low but rose during the perfusion, varying from 0.4 mmol / L (s. d. 0.8) to 32 mmol / L (s. d. 28.2) (Figure 5B). This increase in bilirubin was associated with a reduction of bile flow and bile mud formation (figure 5B). Usually, bile mud made irrigation necessary intermittent biliary tree to allow biliary drainage suitable.

Oxygen consumption showed an initial peak that fell to a valley level in 8 hours where he remained during the rest of the perfusion (figure 5C). The average oxygen consumption at 1 minute for the 5 experiments was 36.2 ml / min (s. d. 3.37) and in 8 hours this was 9.6 ml / min (s. D. 4.06).

In all five experiments there was a good liver preservation without a global variation of the architecture. There were a few areas of pericentral necrosis that was seen in two livers but overall appearances were indicative of a excellent viability Three livers showed areas of a slight septal hemorrhage Four livers showed signs of edema, three Moderate and a severe one. There was sinusoidal congestion present in all the specimens examined, which represented 5-10% of lobes in two cases, 20-25% of lobes in two cases and 40% lobes in the rest of the liver. The dilation of the central vein was present in all but one liver, which It accounted for 25-50% of central veins. In all the Livers examined had a hyperuplasia of Kupffer cells. In four of five livers there was thickened bile present in the bile ducts Two livers had cell infiltrates diffusely mononuclear within the fibrous septum while the three remaining livers did not show them.

Discussion

The technique used for infusion extracorporeal liver according to the present invention could maintain a good function and structure for at least 72 hours in the isolated perfused organ. Liver perfusion isolated has not been described above for periods greater than 24 hours (Neuhaus et al., Int. J. Artif. Organs, 1993; 16: 729-739). Previous researchers normally they put an end to perfusion as a result of progressive acidosis, hyperkalemia and increasing portal pressures. For this reason the invention represents an important advance in the perfusion of liver.

Previous researchers of liver perfusion isolated from large animals have claimed that they have maintained physiological hemodynamic parameters. However, the present inventors have found that if the pumps were controlled to maintain physiological, portal and IVC physiological pressure, blood flow was not maintained at physiological levels. The total hepatic flow was greater and the ratio between portal venous flow and hepatic arterial flow was greater (7: 1) than that described in vivo (3: 1). The increase in blood flow was through the portal vein. A review of the literature does not provide any convincing evidence that a previous researcher has maintained normal hepatic flows and pressures in a synchronized manner. The inventors' work shows that the portal pressure is self-regulated and influenced by the VCI pressure.

It is necessary to indicate the capacity of the liver perfused isolated to maintain base acid balance and level of potassium in this circuit. This disagrees with the experience of others (Mets et al., Journal of Hepatology 1993; 17: 3-9; Adham and others, Transpl. Int., 1997; 10: 299-311) and may be related to a series of factors including the maintenance of the microstructure of the liver and also the provision of a metabolic substrate. Others researchers have found it necessary to incorporate a membrane of dialysis in the circuit to prevent the development of acidosis and hyperkalemia (Schon et al., Transplantation Proceedings 1993; 25: 3239-3243). Like the restoration of normal pH and the maintenance of potassium is a characteristic clinical data of a good function in a newly transplanted liver, this provides other evidence of the excellent function of perfused livers of according to the present invention.

Although it could reasonably be assumed that oxygen consumption is a measure of hepatocyte function, it fell consistently during the course of perfusion. This is in contrast to other indicators of liver function, particularly synthetic activity. However, the initial values obtained for oxygen consumption are related to numbers derived in vivo by a number of authors (Mathisen and Omland, Scand J Gastroent 25: 1265-1273, 1990; Rasmussen et al., Eur J Surg 159: 201-7, 1993; Lindberg and Clowes, Jr., J Surg Research 31: 156-64, 1981; Imamura, Surgery, Gynaecology and Obstetrics 141: 27, 1975; Tygstrup et al., Scan J Gastroent Suppl 9: 131-138 , 1971 and p139-148; Mets et al., Journal of Hepatology 1993; 17: 3-9). In this experimental situation the liver is denervated and is not exposed to the normal hormonal stimuli to which it is subjected in vivo . It is possible that the liver is relatively inactive in this situation that may explain the relatively low level of observed oxygen consumption. In support of this hypothesis, a sharp transient elevation of oxygen consumption was consistently observed when intermittent insulin bolus were added to the circuit. Bile production in this extracorporeal perfusion system reached a maximum of 12.8 ml / hour, approximately half of that observed in vivo . Bile production decreased over time and thickened bile was observed macroscopically in all livers and histologically in four of five experiments. This may reflect a substrate consumption (including bile salts), a loss of hormonal or nervous stimuli and / or inadequate biliary drainage with accumulation of bile mud (although alkaline phosphatase did not increase). Bilirubin serum levels remained low during the first 24 hours of infusion and began to rise concomitantly with the fall in bile production.

Enzymatic indicators of injury Hepatocyte did not rise during infusion. Using the same assay, these enzymes are substantially elevated in a model swine of acute liver failure used by our group. This suggests that there is a good preservation of the viability of hepatocyte despite biliary obstruction and increased bilirubin

The demonstration of protein synthesis provided another test of hepatocellular function. Figures 4A, 4B and 4C show the indication of a continued synthetic function at over 72 hours of infusion. It is interesting to indicate that at it seems the complement production was not regulated by a feedback mechanism in the liver; it seemed to continue at a constant speed accumulating in the plasma of the circuit isolated (figure 4B). The mechanisms that regulate the production of Complement by the liver are still not well understood. Instead, Factor V activity remained at a relatively level constant throughout the perfusion period of 72 hours. The importance of the different models appreciated in the balance of The synthesis and consumption by these two proteins is unclear.

While 3 of 5 livers did not show necrosis, the few areas of necrosis appreciated were of pericentral location, suggesting an ischemic lesion, but this was not enough to cause an increase in the levels of ALT or alkaline phosphatase. There was consistent evidence of edema, sinusoidal congestion and dilation of the central vein. These results may suggest inadequate venous drainage despite the physiological IVC pressures.

After a first perfusion failure in preliminary experiments, prostacyclin was used so routine Thus, although the role of prostacyclin is not has investigated rigorously, we believe that its use is desirable in liver perfusion It is uncertain if it is the activity vasodilator or anti-platelet of prostacyclin which provides the beneficial effects in this situation. Without However, an agent that imitates one or the other of these activities, or the two, may be useful instead of prostacyclin.

Example 2 Extracorporeal perfusion of hDAF transgenic pig livers with human blood

The perfusion apparatus and technique used in Example 1 was used for perfusion of pig livers transgenic for complement regulatory protein, the factor of the acceleration of human decay (hDAF), when it is perfused with healthy and fresh human blood. Three were studied experimental groups: alloperfusions (normal pig livers perfused with pig blood) and xenoperfusions of livers of both unmodified and transgenic hDAF pigs with blood human

Aloperfusion resulted in the maintenance of a good function and histological structure during periods of minus 72 hours Xenoperfusions were also carried out for periods of 72 hours but, unlike aloperfusion, were marked by a progressive decrease in hematocrit of the circulating blood A histological examination showed a necrosis irregular but most lobes were normal. The function Effective liver was demonstrated both in normal infusion and transgenic

Three groups with 5 experiments were studied in each group.

Group A: Aloperfusions, pig livers normal perfused with pork blood.

Group B: Xenoperfusions, pig livers normal (non-transgenic) perfused with human blood.

Group C: Transgenic xenoperfusions, livers of transgenic pigs perfused with human blood.

In each group the same protocol was applied experimental.

Donor pig livers were prepared as in Example 1. Human blood was obtained from voluntary donors and was used in 4 hours of donation.

The perfusion circuit (Figure 1) was used as in Example 1.

The infusion stopped electively at 72 hours or earlier if it was considered that there had been an insufficiency hepatic -pH <7.0, a rapid rise in potassium or an increase of portal pressure. At this time multiple samples were taken of the liver for both immunohistochemical evaluation and staining of hematoxylin and eosin.

Serum samples were analyzed for Complement production by CH50 analysis. The V factor is measured using a prothrombin time based analysis (Organon Teknika MD180). Prothrombin time was also measured. Electrolytes, biochemical liver function tests were measured, creatinine, and urea using standard clinical procedures. Be performed a complete blood count analysis using a counter Coulter (T660) regulated for the measurement of pig blood.

Results

Table 1 below shows the data of flow and pressure for the three groups.

These results have been analyzed using a two-tailed T test (Table 2). None found important difference for portal or total flow between Transgenic and non-transgenic xenoperfusions. However, the alloperfusions showed total and portal flows significantly higher than xenoperfusions (p = 0.05, p = 0.025). Respect both arterial flow and IVC pressure, were appreciated important differences between control xenoperfusions and transgenic (p = 0.05, p = 0.05). Could not show any important difference in blood flow between any of the experimental groups. Although no difference was appreciated important in portal pressure between alloperfusions and transgenic xenoperfusions, important differences were shown between non-transgenic xenoperfusions and both alloperfusions as transgenic xenoperfusions (p = 0.01, p = 0.01).

As a measure of metabolic function the acid-base balance of the circuit was evaluated by pH, bicarbonate and excess base. Excess of base reflects only the metabolic effect of the perfused liver in isolation. In all three groups it was corrected so progressive an initial deep metabolic acidosis. The transgenic xenoperfusions behaved similarly to those alloperfusions (no major difference), while the acid-base was effectively corrected in the non-transgenic xenoperfusions (p = 0.005) (Figure 6A). As a measure Additional liver metabolic function levels were measured Potassium Both xenoperfusion groups showed levels of potassium significantly higher than alloperfusions (p = 0.01, p = 0.05), more marked after 48 hours of infusion (Figure 6B).

Oxygen consumption (ml / hour) for all three Groups are indicated in Table 3 below.

A model consisting of the consumption of oxygen in both xenoperfusion groups with an initial drop followed by a terminal increase; the latter was not appreciated in the alloperfusions

Bile production (ml / hour) in all three Groups are indicated in Figure 7.

Although there was no important difference in the production of bile among the xenoperfusion group, these produced significantly less bile than alloperfusions (p = 0.025, 0.05). Total bilirubin levels rose so progressive in all three groups. Although there were none important difference between xenoperfusions (ending at 138.8 mmol / L and 160.5 mmol / L), the level in the alloperfusion group was significantly lower (32 mmol / L) (p = 0.0005, p = 0.0005) (Figure 7).

While the white blood cell count in both xenoperfusion groups fell to less than 1.0 x 10 9 / L in 1 hour, the white blood cell count of> 1.0 x 10 9 / L is kept up to 52 hours during alloperfusions (not important) (Figure 8B). Platelet count also fell during Normal and transgenic xenoperfusions, the only difference important was between non-transgenic xenoperfusions and the alloperfusions (p = 0.005) (Figure 8A). There was a reduction progressive hematocrit in all three groups (Figure 8C). In 72 hours the terminal hematocrit was 46.9%, 1.9% and 0.3% (alloperfusion, non-transgenic, transgenic). They were shown statistically important differences between all 3 groups (Figure 8C).

Transgenic livers synthesized significantly more urea than non-transgenic livers (p = 0.01) (Figure 9A). The alloperfusion group synthesized significantly less urea than any xenoperfusion group (p = 0.025, p = 0.0005). Proving that this was due to the synthesis instead of hemoconcentration, creatinine levels showed a maximum of 1.7 times in 72 hours of perfusion

The activity of factor V measured in the Aloperfusion was approximately 400%; this remained for all perfusion period (Figure 9B). Both xenoperfusion groups showed initial factor V levels of 100% increasing over time at 200% and 324% respectively. There was none important difference between the xenoperfusion group; It is not appropriate to compare the alloperfusion and xenoperfusion groups since the initial values were different and the analysis requires a Calculation of the area below the curve.

The activity of the complement is shown in the Figure 9C All three groups show an initial decrease of complement activity followed by an increase. At 72 hours, the alloperfusion group had a complement activity of 382% (with respect to the starting level), non-transgenic xenoperfusions 27% and transgenic xenoperfusions 88%. It showed a Statistically important difference between all groups.

Alanine transaminase levels were measured terminal of 51.4 units / L, 115 units / L and 128 units / L in alloperfusion, non-transgenic and transgenic groups, respectively. Although there was no difference statistically important among the xenoperfusion groups, the group of Aloperfusion showed significantly lower levels than any xenoperfusion group (p = 0.0005, 0.005). In all the three groups, alkaline phosphatase levels decreased with the time with transgenic xenoperfusions showing levels significantly lower non-transgenic (39 units / liter versus 28 units / liter at 72 hours) (p = 0.005). It is not appropriate compare aloperfusion with xenoperfusions since the level of Normal alkaline phosphatase in pig blood is twice that of the human blood.

In the transgenic group, necrosis was observed in all livers. In three livers the necrosis was uneven, which represented 10-30% of the livers, in one the necrosis was subcapsular, which represented 5% of the livers and in one there was diffuse necrosis, which represented 80% of the livers. No consistent model of liver injury could be determined. Due to the important necrosis in one of the livers, no other histological analysis was possible. In the remaining four livers, sinusoidal dilation was seen in 3, central dilation in 2, and septal edema in one. Thickened bile was present in the intralobular bile ducts of all four livers. There was endothelitis present in one of the four livers. There was hemorrhage present in the lymphatics of one of the livers. There was hypertrophy of Kupffer cells present in all livers with an important iron Perl stain
intracellular

In the non-transgenic group, bleeding was observed in all livers, but necrosis was clear outside the hemorrhagic areas Hemorrhage involved liver parenchyma in all livers, which implied septal and sinusoidal hemorrhage in four livers, while it was more prominent in the area subcapsular in a liver. All livers showed dilation sinusoidal with red blood cells retained in sinusoids, 2 livers they showed signs of dilatation of the central vein, and four of five livers presented septal edema. None of the livers tube thickened bile. One liver had a slight endothelioma. Existed a very slight Kupffer cell hypertrophy and a minimum Intracellular iron Perl staining in 4 out of five livers.

Overall, only bleeding was seen sinusoidal and connective tissue in non-transgenic livers. In these non-transgenic livers existed relatively little necrosis parenchymal or iron accumulation. Instead, the group transgenic showed more extensive coagulant necrosis than in the groups non-transgenic alloperfusion and iron cells intra-Kupffer was more prominent. Unfortunately, a immunohistochemical analysis of frozen sections of these Experiments was not interpretable.

Discussion

The experiments described in Example 2 demonstrate that the invention is not only effective in alloperfusion (as described in Example 1) but also in a xenoperfusion more demanding.

These experiments give clear proof that pig livers work when they are perfused with blood human It has long been recognized that the liver is relatively protected from damage of preformed antibodies (Collins and others, Transplantation 1994; 58: 1162-1171), rarely has a hyperacute rejection and transplantation of liver is frequently performed in the presence of a cross test of positive lymphocytes (Hathaway et al., Trasplantation 1997; 64: 54-59; Goggins et al., Trasplantation 1996; 62: 1794-1798).

Any use of the term hyperacute rejection requires definition; histological appearances of rejection hyperacute match those of acute vascular rejection (Platt, ASAIO. J. 1992; 38: 8-16). In this way, the diagnosis does not only require histological aspects characteristic (microtrombosis and hemorrhage) but also the absence of organ function at any time.

In this study no hyperacute rejection was observed in transgenic or non-transgenic xenoperfusions. This protection of hyperacute rejection may be a function of the liver itself or the non-physiological nature of the perfusion circuit, in particular the limited volume of blood (and components of the immune effector) or the use of heparin. In this context, it should be noted that hyperacute rejection has been shown in a heparinized ex vivo xenoperfusion heart model (Dunning et al., Eur. J. Cardiothorac. Surg. 1994; 8: 204-206).

In a recent study of ex vivo porcine liver xenoperfusions, the deposition of human antibodies and complement components in collaboration with tissue injury was demonstrated in four hours (Pascher et al., Transplantarion 1997; 64: 384-391). Thus, although the liver is protected from hyperacute rejection, it is still vulnerable to antibody-mediated lesions. Previous attempts at perfusion of swine livers ex vivo in clinic have resulted in a failure that was supposed to be immunologically mediated (Abouna et al., Br. J. Surg. 1968; 55: 862). These infusions were characterized by a progressively increasing portal pressure and subsequent perfusion failure. In contrast, in the present study, both in transgenic and non-transgenic xenoperfusions, portal perfusion pressures remained within physiological limits. The interpretation of histology in this study has been limited by the design of the experiment; Samples were only obtained at the end of the experiment, at which time there was a severe ischemic liver injury secondary to the decrease in hematocrit.

Despite the absence of hyperacute rejection in both groups, it is clear that the introduction of the hDAF transgene has Some important consequences. There are important differences between transgenic and non-transgenic perfused livers regarding portal and IVC pressures, blood flow, acid-base maintenance and synthesis of both the urea as complement. It is therefore assumed that, although it is insufficient to destroy the liver, the immediate reaction complement-mediated immunological initiated sufficient injury as to damage the microcirculation and damage the metabolic function and synthetic It can be argued that this effect is likely, in a closed circuit with limited immunological effector mechanisms, it is an underestimation of the effect that the treatment of a patient in which the liver would be exposed to a volume circulating much higher.

Whereas I respect the parameters exposed above the transgenic livers behaved of similar to aloperfused livers, the analysis of others parameters demonstrated a similar behavior between both groups xenoperfused and important differences of the aloperfundido group. These include some hemodynamic parameters, total flow and portal, potassium levels, bile production and levels of bilirubin, transaminase levels and terminal oxygen consumption. An analysis of bile production and bilirubin levels can be complicated by the considerable differences in the decomposition of red blood cells and suspected complications of biliary mud formation. Also, the reduction of bile salts may be an additional factor in the production of bile. Similarly, potassium levels may reflect differences in the breakdown of red blood cells. He performance of both xenoperfused groups deteriorates towards the end of the 72 hour experiments; this is demonstrated by terminal oxygen consumption values.

This is likely to be, at least in part, a reflection of the decrease in hematocrit that leads to a loss of the oxygen carrying capacity of the blood. This is related to the highest level of transaminase enzymes in these experiments what could be therefore a manifestation of ischemia.

An unexpected result of this study was a progressive reduction of hematocrit, which is perceived in the alloperfusions, but very marked in the non-transgenic group and significantly worse in the transgenic group. Although the damage mechanic is caused by the pump and the circuit (it was noted that the hemolysis occurred when blood circulated in the apparatus of perfusion without a liver), this explains a very small proportion of loss of red blood cells Most of the reduction of hematrocyte in the alloperfusion group can be explained by the dilution, the combination of repeated blood sampling and a constant infusion of other liquids (prostacyclin, nutrition).

Hematocrit estimation as a percentage of primer, correction for dilutional effects, translates into values of: perfusion apparatus alone (93.3%), alloperfusion (85.3%), non-transgenic xenoperfusion (3.3%) and transgenic xenoperfusion (0.5%). The important difference between aloperfusion and both Xenoperfusion groups suggest an important effect of etiology immunological Hypotheses have been made about several possible mechanisms These include the effect of anti-human antibodies pigs eluted from the liver or produced by it. These could be effective by opsonization (which results in the elimination of Kupffer cells) or complement-mediated lysis (human or porcine). The pig supplement could be activated directly at Through the alternative path. Finally the system endothelial reticulum in the liver could extract red blood cells by recognizing species-specific differences or for damage to the red blood cells associated with the pump. The important difference in terminal hematocrits between transgenic and non-transgenic groups is not explained, although they may relate to the improved microcirculation in the livers transgenic

The effect is likely to be particularly marking on the artificial situation of the isolated perfusion circuit and, in fact, a calculation based on the rate of blood cell loss red suggests that a blood transfusion might be required to ratio of less than one unit per day if this system is used in Extracorporeal support of a patient.

Although the initial interpretation of the data from these experiments would suggest that xenoperfused livers not they work significantly as well as livers aloperfundidos, a more rigorous examination suggests that this may be lead to error It is remarkable that liver function, as evaluates by those parameters not directly affected by the decomposition of red blood cells, remains at the same level in xenoperfusions (particularly transgenic) as in the alloperfusions In particular, this is illustrated by the synthesis of Factor V and hemodynamic parameters. The measure of Potassium, hydrogen ions, bilirubin and urea are directly affected by the breakdown of red blood cells. Also they complement levels represent a balance between the production and consumption; a higher level of consumption is inevitable in xenoperfusions.

It is clear that perfused livers provide a metabolic function and are capable of normalizing potassium and acid-base balance; These are essential characteristics of good liver function after liver transplantation. Synthetic liver functions, while potentially having some benefit, can be problematic. The synthesis of high levels of porcine complement may result in injury to human tissues that are not protected by specific complement regulators of appropriate species. However, preliminary data from recent experiments suggest that serum xenoperfusions do not produce fresh human red blood cell lysis in vitro . There is a potential incompatibility between porcine proteins and the human environment (Lawson et al., Transplant. Proc. 1997; 29: 884-885; Reverdiau-Moalic et al., Transplant. Proc. 1996; 28: 643-644). Factor V activity in normal pig serum is maintained at a level four times that of human serum when measured using a human functional analysis. It is not known if this will lead to coagulation abnormalities in the clinical situation. Finally, there is some evidence that an antibody response to porcine proteins can be induced. Under certain circumstances, this may lead to an autoimmune phenomenon (Ortel et al., Am. J. Hematol. 1994; 45: 128-135).

These experiments show that the perfusion of a pig liver with human blood is compatible, at least to medium term, with the function comparable to that obtained using swine blood There is some clear evidence of an effect beneficial of the introduction of the hDAF transgene.

Example 3 Extracorporeal support of pigs in acute liver injury

Acute liver injury was induced in eight pigs not transgenic by previous treatment with oral phenobarbitone for 3 days, intragastric carbon tetrachloride instillation and ligation of the hepatic artery.

Four pigs of the anesthetic were allowed to recover. The parameters of liver function (urea and electrolytes, liver function test, coagulation profile, arterial blood gases and serum ammonia) were controlled as developed acute liver injury. The liver lesion was confirmed from postmortem liver biopsies.

Four pigs remained under general anesthesia for 12-15 hours after ligation of the hepatic artery before connecting to the extracorporeal liver perfusion circuit described above. The cardiovascular status of liver function and arterial blood gases were monitored. In all four experiments the duration of the infusion was limited by technical reasons. Post-mortem samples were taken to assess the degree of liver damage; lung and kidney samples were taken to look for signs of embolic phenomena secondary to perfusion.

The four pigs not treated with infusion Extracorporeal liver survived for 15.5 ± 5.2 hours. Acute liver injury developed in all four pigs: there were massive increases in liver enzymes and a significant elevation of ammonia levels. Histology demonstrated a confluent hepatocellular necrosis with almost no cell left viable liver

The four pigs treated with support extracorporeal to the liver survived for 31, 33, 35 and 52 hours (37.8 ± 9.6 hours), significantly more than non-pigs treated (p <0.005).

The oxygenator failed after 24 hours in the First two infusions. In the two remaining infusions, for which used a different type of oxygenator, a failure ventilatory secondary to mucous plugging in pigs needed interruption of liver perfusion after 22 and 37 hours of infusion.

Immediately before starting the infusion of liver pigs were dying, with extreme tachycardia (160 ± 0 beats per minute), hypotension (80 / 42.5 ± 0 / 3.5 mmHg) and metabolic acidosis (mean pH 7.20 ± 0.12). After an hour of extracorporeal liver perfusion, acidosis was reversed, returning to a physiological margin (pH 7.36 ± 0.08), and the tachycardia improved (127.5 ± 3.5 beats per minute).

After stopping the infusion, both pigs will They deteriorated rapidly and died within an hour (Figure 10).

TABLE 1

Hemodynamic data Alloperfusion Do not transgenic Transgenic (s. d.) (s. d.) (s. d.) Total flow (L / min) 2.01 (0.12) 1.80 (0.27) 1.88 (0.20) Portal flow (L / min) 1.75 (0.18) 1.60 (0.24) 1.58 (0.22) Hepatic arterial flow (mm Hg) 0.237 (0.054) 0.229 (0.094) 0.296 (0,065) IVC pressure (mm Hg) 2.20 (1.04) 2.91 (0.86) 1.64 (0.80) Total pressure (mm Hg) 7.08 (1,2) 11.4 (2.9) 7.23 (2.1) Pressure arterial (mm Hg) 90.3 (2.6) 87.0 (4.1) 89.4 (3.8)

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TABLE 2

Statistical importance of the data hemodynamics Alloperfusion front Front alloperfusion Do not transgenic to non-transgenic to transgenic versus transgenic Flow total 0.05 0.05 NS Flow portal 0.025 0.025 NS Flow arterial NS NS 0.05 Pressure of the VCI NS NS 0.05 Pressure arterial NS NS NS Pressure portal 0.01 NS 0.01

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TABLE 3

Oxygen Consumption (ml O2 / min) Alloperfusion Do not transgenic Transgenic 5 Minutes 19.1 35.7 42.5 level Valley 7.01 10.8 18.8 level terminal 7.01 38.6 64.6

Claims (21)

1. Extracorporeal perfusion procedure of a mammalian organ, the organ being distinct from a heart to which it is allowed to beat, whose procedure includes pumping flow of entry of arterial blood to the organ to maintain pressure physiological and variable flow in the artery of the organ and pump venous outflow from the organ to maintain pressure physiological in the exit vein of the organ, while allowing the organ self-regulates blood flow through itself to Get a physiological arterial flow. 2. Method according to claim 1, characterized in that the organ is selected from the group consisting of liver, kidney, pancreas and small intestine. 3. Method according to claim 2, characterized in that the organ is a liver and the method includes regulating the flow of the portal vein and pumping outflow of blood from the liver to maintain the physiological pressure in the vena cava of the liver, while allowing the liver to self-regulate portal pressure. 4. Method according to claim 3, characterized in that it includes collecting the fluid that is lost from the liver and recirculating the fluid collected back into the liver. 5. Procedure to maintain the viability of a mammalian organ, in which procedure the organ is perfused according to the procedure of any of the claims 1 to 4. 6. Method according to claim 4, which includes maintaining the viability of the organ for at least approximately 72 hours 7. Method according to any one of claims 1 to 6 characterized in that the organ is a liver. 8. Method according to claim 7, characterized in that the liver is porcine. 9. Method according to claim 8, characterized in that the pig liver is genetically modified to express human protein. 10. Method according to claim 7, characterized in that the liver is human. 11. Mammalian liver perfusion apparatus which, when connected to the vena cava, the hepatic artery and the vein portal of a mammalian liver, provides a fluid circuit for blood flow through the liver, in whose circuit there is an outlet channel for connection to the vena cava, a pump Adjustable speed output to maintain physiological pressure in the vena cava, an oxygenator and a heat exchanger, and a branch channel that divides the circuit into input channels for the connection to the hepatic artery and portal vein, the circuit also comprising a device to maintain the physiological pressure in the hepatic artery and physiological flow in the portal vein while allowing a liver, when connected to the apparatus, self-regulate blood flow through the artery and self-regulate the pressure in the portal vein. 12. Apparatus according to claim 11, characterized in that said device includes a container for the collection of excess blood resulting from self-regulation by the flow liver in the hepatic artery. 13. Apparatus according to claim 12, characterized in that the container is for the blood supply to the inlet channel for connection to the portal vein of a liver. 14. Apparatus according to claim 13, characterized in that the container is for the supply of blood under the force of gravity to the portal vein of a liver when connected to the apparatus. 15. Device according to any of the claims 12 to 14, including a channel for collecting the fluid that is lost from the liver and transfer the collected fluid to the container. 16. Apparatus according to any of the claims 11 to 15, which includes connections to join the circulation of a mammal to the circuit. 17. Procedure for driving a mammalian liver perfusion apparatus according to any of the claims 11 to 16 connected to the vena cava, the artery liver and portal vein of a mammalian liver and provide a fluid circuit for blood flow through the liver, including the procedure regulate the pump speed of output to maintain the physiological pressure in the vena cava, regulate the pressure of the inlet channel connected to the hepatic artery to maintain the physiological pressure in the hepatic artery and regulate the flow in the input channel connected to the portal vein to maintain the physiological flow in the portal vein, and allow the liver self-regulates blood flow through the artery and self-regulate the pressure in the portal vein. 18. Method according to claim 17, characterized in that the liver is porcine. 19. Method according to claim 18, characterized in that the pig liver is genetically modified to express human protein. 20. Method according to claim 17, characterized in that the liver is human. 21. Mammalian organ perfusion apparatus suitable for perfusion of a selected mammalian organ from the group consisting of liver, kidney, pancreas and intestine thin and that, the connect to the entrance artery and vein of output of a mammalian organ selected from said group provides a fluid circuit for blood flow through of the organ, in whose circuit there is an output channel for the connection to the exit vein of the organ, an outlet pump of adjustable speed to maintain the physiological pressure in the vein output of the organ, an oxygenator and a heat exchanger, and an input channel for connection to an input artery of the organ, the circuit also comprising a device for maintain physiological pressure and variable flow in the artery of organ input while allowing the organ to self-regulate the blood flow through itself to get the flow physiological arterial